Molecular and cell biological approaches will be used to examine the mechanisms whereby Ca-sensitive adenylyl cyclases (ACs) are targeted to domains of high Ca. Experiments using plasma membrane fractionation procedures, cholesterol depletion and immunoprecipitation will be conducted to address the cellular basis for colocalization of ACs and a variety of Ca-entry channels. The mechanism of high affinity inhibition by Ca of AC5 and AC6 will be addressed initially be deletion mutagenesis. The mechanisms of low afffinity inhibition by Ca and its possible dependence on regulation by Mg will be addressed by site-directed mutagenesis in the C1a region of a number of differently-regulated ACs, exploiting developing information on the crystal structure of adenylyl cyclase.